High-throughput combination assay for studying biofilm formation of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli, the most common cause for urinary tract infections, forms biofilm enhancing its antibiotic resistance. To assess the effects of compounds on biofilm formation of uropathogenic Escherichia coli UMN026 strain, a high-throughput combination assay using resazurin followed by crystal violet staining was optimized for 384-well microplate. Optimized assay parameters included, for example, resazurin and crystal violet concentrations, and incubation time for readouts. For the assay validation, quality parameters Z' factor, coefficient of variation, signal-to-noise, and signal-to-background were calculated. Microplate uniformity, signal variability, edge well effects, and fold shift were also assessed. Finally, a screening with known antibacterial compounds was conducted to evaluate the assay performance. The best conditions found were achieved by using 12 µg/mL resazurin for 150 min and 0.023% crystal violet. This assay was able to detect compounds displaying antibiofilm activity against UMN026 strain at sub-inhibitory concentrations, in terms of metabolic activity and/or biomass.

Evaluation and validation of Biolog OmniLog® system for antibacterial activity assays.

Minimal inhibitory concentration of antimicrobials, determined by the broth microdilution method, requires visual assessment or absorbance measurement using a spectrophotometer. Both procedures are usually performed manually, requiring the presence of an operator to assess the plates at specific time point. To increase the throughput of antimicrobial susceptibility testing, and concurrently convert into an automatic assay, the Biolog OmniLog® system was validated for a new, label-free application using standard 96-well microplates. OmniLog was evaluated for its signal strength to ensure that the signal intensity, detected and measured by the system's camera, was satisfactory. Variability due to the plate location inside the OmniLog incubator, as well as variation between wells, was investigated. Then the system was validated by determining the minimal inhibitory concentration of ciprofloxacin, piperacillin and linezolid against a selected Gram-negative and Gram-positive strains. No significant difference was observed in relation to position of the plates within the system. Plate edge effects were noticeable, thus the edge wells were not included in further experiments. Minimal inhibitory concentration results were comparable to those obtained by conventional protocol as well as to values defined by the Clinical Laboratory Standards Institute or published in the literature.

Fabrication of concave micromirrors for single cell imaging via controlled over-exposure of organically modified ceramics in single step lithography.

This work describes the fabrication of concave micromirrors to improve the sensitivity of fluorescence imaging, for instance, in single cell analysis. A new approach to fabrication of tunable round (concave) cross-sectional shaped microchannels out of the inorganic-organic hybrid polymer, Ormocomp®, via single step optical lithography was developed and validated. The concave micromirrors were implemented by depositing and patterning thin films of aluminum on top of the concave microchannels. The round cross-sectional shape was due to residual layer formation, which is inherent to Ormocomp® upon UV exposure in the proximity mode. We show that it is possible to control the residual layer thickness and thus the curved shape of the microchannel cross-sectional profile and eventually the focal length of the micromirror, by simply adjusting the UV exposure dose and the distance of the proximity gap (to the photomask). In general, an increase in the exposure dose or in the distance of the proximity gap results in a thicker residual layer and thus an increase in the radius of the microchannel curvature. Under constant exposure conditions, the radius of curvature is almost linearly dependent on the microchannel aspect ratio, i.e., the width (here, 20-200 µm) and the depth (here, 15-45 µm). Depending on the focal length, up to 8-fold signal enhancement over uncoated, round Ormocomp® microchannels was achieved in single cell imaging with the help of the converging micromirrors in an epifluorescence microscopy configuration.

Inhibitory effect of dietary phenolic compounds on Chlamydia pneumoniae in cell cultures.

Chlamydial infections are very common worldwide. All chlamydial species have a tendency to cause persistent infections, which have been associated to several chronic diseases including blinding trachoma, infertility and coronary heart disease (CHD). At present, no efficient treatment for the eradication of chronic chlamydial infections exists and, thus, new antichlamydial compounds are urgently needed. This study was designed to screen antichlamydial activity of natural flavonoids and other natural and structurally similar synthetic compounds against Chlamydia pneumoniae in human cell line (HL). HL cells were infected with C. pneumoniae and incubated 72 h with studied compounds. Reduction in the number of inclusions was determined with immunofluorescence staining. In vitro minimum inhibitory concentration was also determined for some of the most active compounds. Thirty seven percentage of the studied compounds (57 in total) were highly active against C. pneumoniae and all the studied compounds were non-toxic to the host cells at studied concentrations. Our study revealed direct antichlamydial effect for selected polyphenolic compounds against C. pneumoniae, in vitro. We also demonstrated the ability of some of the investigated compounds to accumulate inside cells or into cell membranes and cause inhibition, even when present only prior to infection.

Identification of COMT and ErmC inhibitors by using a microplate assay in combination with library focusing by virtual screening.

The paper describes a process of facilitated screening by using a combination of molecular modelling and a 96-well microplate assay for the identification of novel inhibitors of catechol-O-methyltransferase (COMT) and bacteria expressing ErmC. With the help of virtual screening the number of compounds processed in the in vitro screening assay was reduced from over 200,000 to 49. Out of the 49, two structurally very similar compounds were identified as confirmed hits with reasonable activity (IC50 values of 26 and 73 microM) and thus as potential core structures for further drug design and development.

Nanofibrillar cellulose wound dressing in skin graft donor site treatment.

BACKGROUND: Although new therapeutic approaches for burn treatment have made progress, there is still need for better methods to enhance wound healing and recovery especially in severely burned patients. Nanofibrillar cellulose (NFC) has gained attention due to its renewable nature, good biocompatibility and excellent physical properties that are of importance for a range of applications in pharmaceutical and biomedical fields. In the present study, we investigated the potential of a wood based NFC wound dressing in a clinical trial on burn patients. Previously, we have investigated NFC as a topical functionalized wound dressing that contributes to improve wound healing in mice. METHODS: Wood based NFC wound dressing was tested in split-thickness skin graft donor site treatment for nine burn patients in clinical trials at Helsinki Burn Centre. NFC dressing was applied to split thickness skin graft donor sites. The dressing gradually dehydrated and attached to donor site during the first days. During the clinical trials, physical and mechanical properties of NFC wound dressing were optimized by changing its composition. From patient 5 forward, NFC dressing was compared to commercial lactocapromer dressing, Suprathel® (PMI Polymedics, Germany). RESULTS: Epithelialization of the NFC dressing-covered donor site was faster in comparison to Suprathel®. Healthy epithelialized skin was revealed under the detached NFC dressing. NFC dressing self-detached after 11-21days for patients 1-9, while Suprathel® self-detached after 16-28days for patients 5-9. In comparison studies with patients 5-9, NFC dressing self-detached on average 4days earlier compared with Suprathel®. Lower NFC content in the material was evaluated to influence the enhanced pliability of the dressing and attachment to the wound bed. No allergic reaction or inflammatory response to NFC was observed. NFC dressing did not cause more pain for patients than the traditional methods to treat the skin graft donor sites. CONCLUSION: Based on the preliminary clinical data, NFC dressing seems to be promising for skin graft donor site treatment since it is biocompatible, attaches easily to wound bed, and remains in place until donor site has renewed. It also detaches from the epithelialized skin by itself.

A rapid screening method for detecting active compounds against erythromycin-resistant bacterial strains of Finnish origin.

A rapid and simple microdilution technique on 96-well microplate based on turbidimetry was optimized and validated for screening of antimicrobial activity against erythromycin-resistant bacterial strains of Streptococcus pyogenes and Staphylococcus simulans isolated from Finnish patients. Using S. pyogenes ATCC 12351 as reference strain the developed method was evaluated by reproducibility measurements and using parameters typically employed for screening methods, i.e. signal-to-background, signal-to-noise and a screening-window coefficient, the Z' factor. The method was further used for screening a group of natural compounds and their synthetic derivatives against resistant bacterial strains. Of these, octyl and dodecyl gallates, and usnic and ursolic acids were the most active. The described method is a rapid, homogeneous, cost-effective and easy-to-perform system for screening of new potential antimicrobial agents in drug discovery.

Effects of simple aromatic compounds and flavonoids on Ca2+ fluxes in rat pituitary GH(4)C(1) cells.

The biological activity of phenolic compounds from plants is well documented in vitro, but little is known about the possible effect of simple aromatic compounds and flavonoids on voltage-operated Ca2+ channels (VOCCs). In pituitary cells, several intracellular pathways may regulate the activity of VOCCs. In this study, we investigated the effect of nine phenylpropanes and metanes, and 20 flavonoids on high K(+)-induced 45Ca2+ entry in clonal rat pituitary GH(4)C(1) cells. At the highest dose tested (20 microg/ml), flavone (a flavone) inhibited 45Ca2+ entry by 63.5%, naringenin (a flavanone) by 56.3% and genistein (an isoflavone) by 54.6%. The phenylmetane derivative octyl gallate was the most potent compound tested, with an IC(50) value of 15.0 microg/ml. The IC(50) value for the reference compound verapamil hydrochloride was 3.0 microg/ml. In sharp contrast to the above, the flavonols quercetin and morin potentiated 45Ca2+ entry. At 20 microg/ml, quercetin increased 45Ca2+ entry by 54.1% and morin by 48.0%. Quercetin increased the cellular cAMP content in a concentration-dependent manner. H 89, an inhibitor of protein kinase A, inhibited the effect of quercetin on 45Ca2+ entry. The results thus suggest that the effect of quercetin is the result of a protein kinase A-mediated activation of VOCCs. Quercetin induced a rapid and marked increase in both the transient (143.1+/-4.2%) and delayed (198.8+/-10.0%) Ca2+ currents, measured by the whole cell patch clamp technique. The onset of the inhibitory effect of octyl gallate was slow, but resulted in an almost complete inhibition of both Ca2+ currents.

In vitro assay for human toxicity of cereulide, the emetic mitochondrial toxin produced by food poisoning Bacillus cereus.

The in vitro boar spermatozoon test was compared with the LC ion trap MS analysis for measuring the cereulide content of a pasta dish, implemented in serious emetic food poisoning caused by Bacillus cereus. Both assays showed that the poisonous food contained approximately 1.6 microg of cereulide g(-1) implying the toxic dose in human as < or =8 microg kg(-1) body weight. The threshold concentration of cereulide provoking visible mitochondrial damage in boar sperm exposed in vitro was 2 ng of cereulide ml(-1) of extended boar sperm. The same threshold value was found for cereulide extracted from the food and from the cultured bacteria. This shows that other constituents of the food did not enhance or mask the effects of cereulide. Exposure of four human cell lines (HeLa, Caco-2, Calu-3 and Paju) to cereulide showed that the threshold concentration for the loss of mitochondrial membrane potential in human cells was similar to that observed in boar sperm. Human cells and boar sperm were equally sensitive to cereulide. The results show that boar spermatozoan assay is useful for detecting cereulide concentrations toxic to humans. Spermatozoa in commercially available extended fresh boar and cryopreserved bull semen were compared, boar sperms were 100 times more sensitive to cereulide than bull sperms.

Effects of the aqueous extract of Bryothamnion triquetrum on chemical hypoxia and aglycemia-induced damage in GT1-7 mouse hypothalamic immortalized cells.

The neuroprotective ability of the aqueous crude extract of Bryothamnion triquetrum (S. G. Gmelin) Howe and its cinnamic acids was studied in GT1-7 cells exposed to the combination of chemical hypoxia (KCN 3 mM) and aglycemia conditions. These ischemia-like conditions provoked acute and delayed cytotoxicity in GT1-7 cells if extended for more than 90 min. The extract was able to protect from the cell death produced by severe (180 min) chemical hypoxia/aglycemia insult, which cannot be related to its glucose content, and also reduced the cytotoxicity and early production of free radicals produced by mild (105 min) insult. Results showed that some of these protective effects of the extract are partially related to the presence of ferulic acid. The data additionally suggest that neuroprotection exerted by the extract is related to its ability to reduce free-radical generation by mechanisms different from the direct scavenging of the radical entities.

Aging in Pinus sylvestris L. seeds: changes in viability and lipids.

Aging of Scots pine seeds (Pinus sylvestris L.) leads to changes in seed quality, such as loss of germinability, delayed growth and abnormality in developing seedlings. The knowledge of biochemical changes responsible for these aging processes is plentiful in some seeds, which are of world-wide interest, but for pine seeds these studies are rare. The aim of the present study, was to analyse pine seeds of varying ages in order to identify biochemical changes occurring in aged pine seeds, and to see if a correlation existed between these results and traditionally used seed-quality parameters, such as germinability and electrolyte leakage.